Vasopressin is the major antidiuretic hormone involved in the regulation of water reabsorption by the mammalian kidney. It induces a sudden and dramatic increase in apical plasma membrane water permeability of principal cells in the kidney collecting duct that allows a hypertonic urine to be produced. By analogy with studies on the toad urinary bladder, it is believed that the cell biological mode of action of vasopressin in the kidney is to induce the exocytotic fusion of cytoplasmic vesicles containing water channels with the plasma membrane. The insertion of these channels renders the apical membrane permeable to water; the reverse process occurs upon hormonal withdrawal, and water channels are removed from the plasma membrane in endocytotic vesicles. We have shown that this endocytosis occurs via clathrin coated pits, that the internalized endosomes do indeed contain water channels, and that these endosomes represent a specialized endosomal compartment that does not acidify, thus distinguishing them from generic endosomes in other cell types. Nevertheless, key questions remain unanswered, including a) what is the identity of the water channel, b) how are water channels inserted into the plasma membrane in principal cells, c) is there any cross-talk between basolateral endosomes and apical endosomes in principal cells, or are they two completely distinct pathways? Our present specific aims will directly address these questions by, 1) purifying and characterizing the protein content and the functional attributes of the endosomes that internalize vasopressin-sensitive water channels, 2) using a combination of techniques together with combined in vivo-in vitro studies to examine vesicle recycling in principal cells, with special emphasis on identifying the vesicles involved in vasopressin-induced exocytosis of water channels, 3) examining intracellular pathways of apical- and basolaterally-derived endosomes in principal cells with different fluorescent tracers, antibodies and functional assays in normal and Brattleboro rats. Because the basolateral membrane of principal cells has a high, vasopressin-independent intrinsic permeability to water, it is important to determine if this membrane could, by transcytosis of basolateral vesicles, enter the apical recycling pathway and contribute water permeable membrane to this pathway. As we have done in the past, we will continue to exploit a system that contributes not only to an understanding of renal physiology, but also to more general mechanisms underlying the generation, maintenance and modulation of polarized function in epithelial cells.